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C Sasakawa

Publications and source records attributed to C Sasakawa.

At least 19 recordsLinked to original sources
PubMed2003

Kinetics of v-src-induced epithelial-mesenchymal transition in developing glandular stomach.

The oncogene function in primary epithelial cells is largely unclear. Recombination organ cultures in combination with the stable and transient gene transfer techniques by retrovirus and electroporation, respectively, enable us to transfer oncogenes specifically into primary epithelial cells of the developing avian glandular stomach (proventriculus). In this system, the epithelium and mesenchyme are mutually dependent on each other for their growth and differentiation. We report here that either stable or transient expression of v-src in the epithelium causes budding and migration of epithelial cells into mesenchyme. In response to the transient expression of v-Src or a constitutive active mutant of MEK, we observed immediate downregulation of the Sonic hedgehog gene and subsequent elimination of E-cadherine expression in migrating cells, suggesting the involvement of MAP kinase signaling pathway in these processes. v-src-expressing cells that were retained in the epithelium underwent apoptosis (anoikis) and detached from the culture. Continuous expression of v-src by, for example, Rous sarcoma virus (RSV) was required for the epithelial cells to acquire the ability to express type I collagen and fibronectin genes (mesenchymal markers), and finally to establish the epithelial-mesenchymal transition. These observations would partly explain why RSV does not apparently cause carcinoma formation, but induces sarcomas exclusively.

Animals↗
PubMed2001

Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure.

Enteropathogenic Escherichia coli (EPEC) secretes several Esp proteins via the type III secretion system (secreton). EspA, EspB, and EspD are required for translocation of the effector proteins into host cells, in which EspB and EspD are thought to form a pore in the host membrane. Recent study has shown that EspA forms a filamentous structure that assembles as a physical bridge between bacteria and host cell surfaces, which then functions as a conduit for the translocation of bacterial effectors into host cells. To investigate the supermolecular structure of the type III secreton in EPEC, we partially purified it from the bacteria membrane and observed it via transmission electron microscopy. The EPEC type III secreton was composed of a basal body and a needle part and was similar to those of Salmonella and Shigella, except for a sheath-like structure at the tip of the needle. The length of sheath-like structures varied; it extended more than 600 nm and was 10 times longer than the Shigella needle part. The putative major needle component, EscF, was required for both secretion of Esp proteins and needle complex formation. Interestingly, elongation of the sheath-like structure was observed under constitutive expression of EspA but not of EscF. Furthermore, the transmission electron microscopy view with immunogold labeled anti-EspA antibodies clearly showed that EspA is a component of the sheath-like structure. This study revealed, to our knowledge for the first time, the supermolecular structure of the EPEC type III secreton and its direct association with the EspA-sheath-like structure.

Amino Acid Sequence↗
PubMed2001

Shigella protein IpaH(9.8) is secreted from bacteria within mammalian cells and transported to the nucleus.

Various pathogenic bacteria such as Shigella deliver effector proteins into mammalian cells via the type III secretion system. The delivered Shigella effectors have been shown to variously affect host functions required for efficient bacterial internalization into the cells. In the present study, we investigated the IpaH proteins for their ability to be secreted via the type III secretion system and their fate in mammalian cells. Upon incubation in a medium containing Congo red, the bacteria secrete IpaH into the medium, but secretion of IpaH occurs later than that of IpaBCD. Immunofluorescence microscopy indicated that IpaH(9.8) is secreted from intracellular bacteria and transported into the nucleus. On microinjection of the protein, intracellular IpaH(9.8) is accumulated at one place around the nucleus and transported into the nucleus. This movement seems to be dependent on the microtubule network, since nuclear accumulation of IpaH(9.8) is inhibited in cells treated with microtubule-destabilizing agents. In nuclear import assay, IpaH(9.8) was efficiently transported into the nucleus, which was completely blocked by treatment with wheat germ agglutinin. The nuclear transport of IpaH(9.8) does not depend on host cytosolic factors but is partially dependent on ATP/GTP, suggesting that, like beta-catenin, IpaH(9.8) secreted from intracellular Shigella can be transported into the nucleus.

Amino Acid Sequence↗
PubMed2001

Shigella invasion of macrophage requires the insertion of IpaC into the host plasma membrane. Functional analysis of IpaC.

Shigella infects residential macrophages via the M cell entry, after which the pathogen induces macrophage cell death. The bacterial strategy of macrophage infection, however, remains largely speculative. Wild type Shigella flexneri (YSH6000) invaded macrophages more efficiently than the noninvasive mutants, where YSH6000 induced large scale lamellipodial extension including ruffle formation around the bacteria. When macrophages were infected with the noninvasive ipaC mutant, the invasiveness and induction of membrane extension were dramatically reduced as compared with that of YSH6000. J774 macrophages infected with YSH6000 showed tyrosine phosphorylation of several proteins including paxillin and c-Cbl, and this pattern was distinctive from those stimulated by Salmonella typhimurium or phorbol ester. Upon addition of IpaC into the external medium of macrophages, membrane extensions were rapidly induced, and this promoted uptake of Escherichia coli. The exogenously added IpaC was found to be integrated into the host cell membrane as detected by immunostaining. The IpaC domain required for the induction of membrane extension from J774 was narrowed down within the region of residues 117-169, which contains a putative membrane-spanning sequence. Our data indicate that Shigella directs its own entry into macrophages, and the IpaC domain which is required for the association with its host membrane is crucial.

Amino Acid Sequence↗
PubMed2001

Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12.

Escherichia coli O157:H7 is a major food-borne infectious pathogen that causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Here we report the complete chromosome sequence of an O157:H7 strain isolated from the Sakai outbreak, and the results of genomic comparison with a benign laboratory strain, K-12 MG1655. The chromosome is 5.5 Mb in size, 859 Kb larger than that of K-12. We identified a 4.1-Mb sequence highly conserved between the two strains, which may represent the fundamental backbone of the E. coli chromosome. The remaining 1.4-Mb sequence comprises of O157:H7-specific sequences, most of which are horizontally transferred foreign DNAs. The predominant roles of bacteriophages in the emergence of O157:H7 is evident by the presence of 24 prophages and prophage-like elements that occupy more than half of the O157:H7-specific sequences. The O157:H7 chromosome encodes 1632 proteins and 20 tRNAs that are not present in K-12. Among these, at least 131 proteins are assumed to have virulence-related functions. Genome-wide codon usage analysis suggested that the O157:H7-specific tRNAs are involved in the efficient expression of the strain-specific genes. A complete set of the genes specific to O157:H7 presented here sheds new insight into the pathogenicity and the physiology of O157:H7, and will open a way to fully understand the molecular mechanisms underlying the O157:H7 infection.

Bacterial Proteins↗
PubMed2001

Role of bundle-forming pilus of enteropathogenic Escherichia coli in host cell adherence and in microcolony development.

Enteropathogenic Escherichia coli (EPEC) adheres to epithelial cells and forms microcolonies in localized areas. Bundle-forming pili (BFP) are necessary for autoaggregation and the formation of microcolonies. In this study, we show that BFP, expressed by EPEC on epithelial cells, disappeared with the expansion of the microcolony. Bacterial dispersal and the release of BFP from the EPEC aggregates were induced by contact with host cellular membrane extract. In addition, BFP-expressing EPEC adhered directly to cell surfaces, in preference to attaching to pre-formed microcolonies on the cells. These results suggested that BFP mediate the initial attachment of EPEC through direct interaction with the host cell rather than through the recruitment of unattached bacteria to microcolonies on the cell.

Bacterial Adhesion↗
PubMed2001

Enhanced anti-Shigella activity of erythromycin supplemented with sulfadiazine.

Sulfadiazine enhanced the anti-Shigella activity of erythromycin. Erythromycin passes through the type III secretion apparatus and suppresses the growth of invasive Shigella organisms. Sulfadiazine enhanced this effect at the concentration under minimum inhibitory concentration and it came from not only the folate-inhibiting activity but also from a new function. It has proved that sulfadiazine stimulated type III secretion in Shigella as determined from the secretion of the pathogenic protein IpaB. As Congo red induced secretion of Ipa proteins and uptake of erythromycin through the type III secretion gate, sulfadiazine which is similar to Congo red in chemical structure may induce the uptake in the same way.

Animals↗
PubMed2001

toxB gene on pO157 of enterohemorrhagic Escherichia coli O157:H7 is required for full epithelial cell adherence phenotype.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is critical for initiation of a bacterial infection. An in vitro infection study previously indicated that EHEC bacteria initially adhere diffusely and then proliferate to develop MC, a process that is mediated by various secreted proteins, such as EspA, EspB, EspD, Tir, and intimin, as well as other putative adherence factors. In the present study, we investigated the role of a large 93-kb plasmid (pO157) in the adherence of O157:H7 (O157Sakai) and found the toxB gene to be involved in the full adherence phenotype. A pO157-cured strain of O157Sakai (O157Cu) developed microcolonies on Caco-2 cells; however, the number of microcolonies was lower than that of O157Sakai, as were the production and secretion levels of EspA, EspB, and Tir. Introduction of a mini-pO157 plasmid (pIC37) composed of the toxB and ori regions restored full adherence capacity to O157Cu, including production and secretion of the proteins. In contrast, introduction of a pO157 mutant possessing toxB::Km into O157Cu could not restore the full adherence phenotype. Expression of truncated versions of His-tagged ToxB also promoted EspB production and/or secretion by O157Cu. These results suggest that ToxB contributes to the adherence of EHEC to epithelial cells through promotion of the production and/or secretion of type III secreted proteins.

Bacterial Adhesion↗
PubMed2001

EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri.

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic Escherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with Shigella flexneri protein VirA, and the cloned espG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPEC espG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned orf3 gene could also rescue invasion in a Shigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.

Amino Acid Sequence↗
PubMed2001

Structural and functional characterization of IS679 and IS66-family elements.

A new insertion sequence (IS) element, IS679 (2,704 bp in length), has been identified in plasmid pB171 of enteropathogenic Escherichia coli B171. IS679 has imperfect 25-bp terminal inverted repeats (IRs) and three open reading frames (ORFs) (here called tnpA, tnpB, and tnpC). A plasmid carrying a composite transposon (Tn679) with the kanamycin resistance gene flanked by an intact IS679 sequence and an IS679 fragment with only IRR (IR on the right) was constructed to clarify the transposition activity of IS679. A transposition assay done with a mating system showed that Tn679 could transpose at a high frequency to the F plasmid derivative used as the target. On transposition, Tn679 duplicated an 8-bp sequence at the target site. Tn679 derivatives with a deletion in each ORF of IS679 did not transpose, finding indicative that all three IS679 ORFs are essential for transposition. The tnpA and tnpC products appear to have the amino acid sequence motif characteristic of most transposases. A homology search of the databases found that a total of 25 elements homologous to IS679 are present in Agrobacterium, Escherichia, Rhizobium, Pseudomonas, and Vibrio spp., providing evidence that the elements are widespread in gram-negative bacteria. We found that these elements belong to the IS66 family, as do other elements, including nine not previously reported. Almost all of the elements have IRs similar to those in IS679 and, like IS679, most appear to have duplicated an 8-bp sequence at the target site on transposition. These elements have three ORFs corresponding to those in IS679, but many have a mutation(s) in an ORF(s). In almost all of the elements, tnpB is located in the -1 frame relative to tnpA, such that the initiation codon of tnpB overlaps the TGA termination codon of tnpA. In contrast, tnpC, separated from tnpB by a space of ca. 20 bp, is located in any one of three frames relative to tnpB. No common structural features were found around the intergenic regions, indicating that the three ORFs are expressed by translational coupling but not by translational frameshifting.

Amino Acid Sequence↗
PubMed2001

Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium.

Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium. We compared their genetic and pathological features to reveal the relationship between these two bacteria. To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested. Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed. MPEC strains and C. rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences. All results from biochemical reactions and the mouse strain susceptibility tests were identical. It is proposed that MPEC should be reclassified as C. rodentium.

Animals↗
PubMed2000

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Amino Acid Sequence↗
PubMed2000

Profilin is required for sustaining efficient intra- and intercellular spreading of Shigella flexneri.

The ability of Shigella to mediate actin-based motility within the host cell is a prominent pathogenic feature of bacillary dysentery. The ability is dependent on the interaction of VirG with neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn mediates recruitment of Arp2/3 complex and several actin-related proteins. In the present study, we show that profilin I is essential to the rapid movement of Shigella in epithelial cells, for which the capacity of profilin to interact with G-actin and N-WASP is critical. In COS-7 cells overexpressing either mutated profilin H119E, which failed to bind G-actin, or H133S, which is unable to interact with poly-l-proline, Shigella motility was significantly inhibited. Similarly, depletion of profilin from Xenopus egg extracts resulted in a decrease in bacterial motility that was completely rescued by adding back profilin I but not H119E or H133S. In COS-7 cells overexpressing a N-WASP mutant lacking the proline-rich domain (Deltap) unable to interact with profilin, the actin tail formation of intracellular Shigella was inhibited. In N-WASP-depleted extracts, addition of Deltap but not full-length N-WASP was unable to restore the bacterial motility. Furthermore, in a plaque formation assay with Madin-Darby canine kidney cell monolayers infected by Shigella, Madin-Darby canine kidney cells stably expressing H119E, H133S, or Deltap reduced the bacterial cell-to-cell spreading. These results indicate that profilin I associated with N-WASP is an essential host factor for sustaining efficient intra- and intercellular spreading of Shigella.

Actins↗
PubMed2000

Supramolecular structure of the Shigella type III secretion machinery: the needle part is changeable in length and essential for delivery of effectors.

We investigated the supramolecular structure of the SHIGELLA: type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 +/- 3.3 and 31.6 +/- 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F(1)-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete effector proteins. Upon overexpression of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle.

Adhesins, Bacterial↗
PubMed2000

Rho family GTPase Cdc42 is essential for the actin-based motility of Shigella in mammalian cells.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.

3T3 Cells↗
PubMed2000

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

Amino Acid Sequence↗